Abstract Gastrointestinal (GI) complications are a significant clinical and economic burden in patients with Parkinson?s disease (PD). Specifically, constipation is responsible for the most drastic decrease in the quality of life for 24- 63% of PD patients. Yet, due to the lack of knowledge of the mechanisms underlying this symptom, there haven?t been successful or long-term therapies. It has been well established that aggregated forms of ?- synuclein (?-syn), a presynaptic terminal protein identified to be the pathological marker in PD, is observed diversely through the enteric nervous system (ENS) of the GI tract in PD patients. ?-syn aggregation within the ENS as well as constipation precede the motor deficits in PD by up to 20 years. Whether this protein is interfering with neurotransmission in the ENS is currently unknown. Our preliminary data in recombinant adeno-associated virus with A53T point mutation targeted to enteric neurons in the mouse model found reduced colonic propulsion suggesting that ?-syn maybe disrupting cholinergic neurotransmission. The long- term objective of this project is to determine whether ?-syn aggregation in the colonic myenteric plexus impairs ENS cholinergic neuromuscular and synaptic transmission causing abnormal colonic propulsion. To achieve this project objective, Aim 1 will determine the expression of ectopic ?-syn in the myenteric neurons of the colon as this will provide us mechanistic data on the potential interactions between the protein and neurotransmitter systems. In Aim 2 we will use in vivo and ex vivo paradigms to determine whether ?-syn overexpression disrupts propulsive colonic motility by addressing colonic propulsion and colonic contractions and relaxations. Aim 3 will use optogenetics to identify the effect of ?-syn overexpression on cholinergic synaptic and neuromuscular transmission in the myenteric plexus. This proposed research will be the first to directly study cholinergic neurotransmission within the ENS in the presence of ?-syn aggregates. Moreover, we aim to identify novel targets that may initiate new drug discoveries in treating constipation involved in PD.